Created by: Jack Renner 

       Pseudomonas aeruginosa is a bacterium that uses quorum sensing to communicate behaviors for a large population (1). These signals are often associated with increases in the population density to a sufficient point that allows the bacteria to attack a host (1). The LasR receptor (PDB ID: 6D6P) receives these intercellular signals, and promotes the transcription of virulence factors necessary for infection (1,2). Once LasR has bound its homoserine lactone ligand (AHL), it then binds DNA and alters transcription of other proteins, leading to proliferation and host infection by the bacterium (1,2). Studying the binding site of LasR in order to determine possible antagonists could serve to reduce the virulence of Pseudomonas aeruginosa, thus preventing many nosocomial infections that the bacteria is responsible for (3). 

       The LasR receptor is a monomeric protein with 170 residues (4). The weight of the isolatable dimer is 36531.93 Da, and the isoelectric point of the monomer is at pH 6.09, as determined by the ExPASy database (4,5). The LasR receptor is comprised of two domains, one ligand-binding and one DNA binding domain (6). The ligand binding domain has a five-stranded antiparallel β sheet with three α helices on each side (6). The ligand pocket is located between the β sheet and helices α3, α4, and α5 (6). The primary ligand for LasR is N-{[3,5-dibromo-2- (methoxymethoxy)phenyl]methyl}-2-nitrobenzamide. This ligand is among the family of homoserine lactones, and there are many similar molecules that have the ability to bind in the LasR binding domain, but their affinity to the pocket prevents them from activating LasR (1). 

       Loop L3 of LasR (residues Leu-40 - Phe-51) forms a sort of flap over the ligand pocket, preventing the ligand from interacting with the solvent and environment (6). Within the binding pocket, residues Tyr-56, Trp-60, Asp-73, and Ser-129 all form hydrogen bonds with the polar head group of the homoserine lactone ligand (6). Residues Leu-36, Leu-40, Ile-52, Val-76, and Leu-125 all have van der Waals interactions with the hydrophobic tail of the homoserine lactone (6). Tyr-47 is essential to the conformation change of loop L3, as it stabilizes the residue in the hydrophobic environment of the hydrocarbon chain (6). This is important to note because changing the length of this hydrocarbon chain can alter the favorable interaction, reducing the conformation change, and causing the ligand to be more exposed to the solvent (6). When the ligand is more exposed to the solvent, it is more susceptible to detach due to less favorable interactions with LasR. This is where the difficulty lies in creating antagonists for LasR; the ligand must cause a conformational change to remain in the pocket, but also prevent LasR from promoting transcription (6). 

       Because of this instability in the ligand pocket, researchers are studying agonists to LasR in hopes to better understand the ligand binding domain, leading to more success in creating effective antagonists. Currently, researchers are using variations of a triphenyl compound to mimic some of interactions of homoserine lactones in order to cause the proper conformational changes, previously only thought possible with a homoserine lactone derivative (1). Among these compounds, several have resulted in 98% LasR activity when bound, and resulted in conformation changes that covered over 650Ų of the pocket. These triphenyl derivative agonists could serve to be a structural basis for the first synthetic antagonists against LasR. 

       The induced conformation of LasR is generally thought to be required for transcriptional changes to occur. The mechanism by which is happens is proposed to be direct DNA binding or interactions with RNA polymerase, but is currently unknown (3). Binding its ligand causes an increase in stability of monomeric LasR, and leads to the formation of a homodimer of two AHL-stabilized LasR molecules (1). The LasR homodimer is formed from polar and nonpolar interactions between helix α6 and two loop regions of the adjacent monomer (6). These homodimers tend to aggregate into pairs in solution, forming a tetramer. This is thought to be the complex that interacts with the DNA or RNA polymerase to promote transcription (1,4). 

       The Basic Local Alignment Search Tool (BLAST) compares the sequence of proteins and provides an E value to represent how similar two sequences are. Lower E values indicate more similarity between the sequences (i.e, less amino acid differences), and E values below 0.05 indicate significant similarity (7). The Dali server compares the tertiary (3D) structure of proteins, and assigns a Z score to measure similarity. Z scores above 2 are considered and indication of significant structural similarity (8). QscR (PDB ID: 3SZT) from Pseudomonas aeruginosa is also involved in quorum sensing in bacteria. QscR operates via the same mechanism of LasR, and is considered to be within the same family of LuxR receptor proteins (2,9). QscR has an E value of 0.11, indicating that its sequence is not completely similar to that of LasR, yet still has a very similar function (7). QscR was given a Z score of 18.6, indicating significant structural similarity (8). 

       QscR's monomer has 237 residues, and dimerizes once bound to its ligand. QscR, like LasR, has a secondary structure consisting of a five-stranded antiparallel β sheet with 3 α helices on each side (4,9). The non ligand binding region in QscR, however, is elongated, and thus the interaction between monomers is different. The dimer in QscR is symmetrical between residues Glu-84 and Lys-121. Unlike LasR, however, QscR does not form a tetramer primarily due to the asymmetric nature of its monomer. This only affects the mechanism by which QscR associates with other proteins to influence transcription. 

       QscR is primarily used to research possible ways to disrupt the dimerization of these LuxR type proteins. These proteins are classified by their ligands being homoserine lactone variants, generally multimerizing once bound, and influencing transcription leading to increased bacterial virulence. Data has shown that disruption of the symmetric interactions between monomers of QscR resulted in dramatic decreases in protein activity. Because many LuxR type receptors' functions rely on these interactions, QscR is a suitable model for beginning to understand how to disrupt these interactions. This could also prove to be a useful approach to reducing quorum sensing in Pseudomonas aeruginosa (9). QscR's ligand binding site is also specific to the hydrocarbon chain of the homoserine lactone, and the conformational change associated with the stability increase from its interaction is essential to its function, as it is in LasR (1,9). This interaction reinforces the notion that any potential antagonists to these receptors must provide a similar stability increase for the protein to retain the substituted ligand. Although the specific mechanism of influencing transcription is unknown, QscR, like LasR, is thought to either bind directly to the DNA strand, or to a replication enzyme involved in RNA synthesis. 

 

       The function of LasR appears to be primarily affected by the conformation shift of the L3 loop when bound to a ligand. The native homoserine lactone for LasR interacts with the Tyr-47 residue on L3. Other potential ligands must cause the L3 loop to shift to the closed conformation in order to allow LasR to function properly, but also not release the ligand. In the open conformation, the ligand is susceptible to removal due because of less interactions and no spacial hindrance (1). Research of LasR and other LuxR type receptors is of vital importance to the medical industry as potential inhibition points for common nosocomial infections resulting from quorum sensing leading to increases in bacterial virulence (3).